EST-SSR (expressed sequence tag-simple sequence repeat) marker combination and application thereof in polygonatum plant identification

Abstract

The invention discloses an EST-SSR (expressed sequence tag-simple sequence repeat) molecular marker combination and application of the EST-SSR molecular marker combination in identification of polygonatum plants, and belongs to the technical field of plant molecular biology. 49 pairs of high-polymorphism EST-SSR marker primers are developed on the basis of rhizome transcriptome joint data of three medicinal polygonatum sibiricum species, namely polygonatum kingianum, polygonatum kingianum and polygonatum cyrtonema, and 9 pairs of core marker primers are screened from the 49 pairs of high-polymorphism EST-SSR marker primers. The invention further provides a kit containing the marker combination and an identification method, a fluorescent capillary electrophoresis detection technology is adopted, six sibling species of polygonatum kingianum, polygonatum kingianum, polygonatum cyrtonema, polygonatum kingianum, polygonatum kingianum and Hubei polygonatum kingianum can be accurately distinguished at a time, and intraspecific variation of different geographical populations of polygonatum kingianum can be effectively analyzed. The marker combination is high in universality, high in polymorphism, large in detection flux and accurate and reliable in result, and an efficient molecular tool is provided for polygonatum germplasm resource identification, genetic diversity analysis and molecular assisted breeding.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically involving an EST-SSR molecular marker primer combination developed based on the transcriptome sequence of medicinal Polygonatum, and its application in the identification of Polygonatum germplasm resources, genetic diversity analysis and molecular-assisted breeding. Background Technology

[0002] Polygonati Rhizoma is a traditional Chinese medicinal herb used in both food and medicine. It originates from various plants in the genus *Polygonatum* of the Liliaceae family, including *Polygonatum sibiricum*, *Polygonatum kingianum*, *Polygonatum cyrtonema*, *Polygonatum kingianum var. grandifolium*, and *Polygonatum cirrhifolium*. These plants are widely distributed and contain various active ingredients such as polysaccharides, saponins, and flavonoids. They possess traditional medicinal properties such as tonifying the middle energizer, strengthening the spleen and lungs, and benefiting the kidneys, as well as modern pharmacological effects such as lowering blood sugar, regulating immunity, and combating fatigue. Market demand for this herb is steadily increasing.

[0003] However, the morphological identification characteristics of plants in the genus *Polygonatum* are complex, with unstable phenotypic traits such as leaf arrangement and rhizome morphology, and significant interspecific overlap, making them prone to confusion, especially during the off-season or when identification is based on the morphology of medicinal materials. This phenomenon of "synonyms" or "synonyms" seriously hinders the standardization of the *Polygonatum* industry and the breeding of high-quality germplasm. Therefore, developing molecular marker technologies that can accurately and efficiently identify *Polygonatum* species and their intraspecific variations is of great significance for germplasm resource conservation, variety identification, and genetic breeding.

[0004] Simple sequence repeat (SSR) markers have become an ideal tool for genetic analysis due to their high polymorphism, codominance, and good reproducibility. Among them, EST-SSR markers derived from expressed sequence tags are located in the gene transcription region, may be associated with functional traits, and have higher universality and conservation among closely related species.

[0005] Existing SSR marker technologies suffer from limitations such as a limited number of markers, incomplete species coverage, low resolution of intraspecific variation, and outdated detection techniques, making it difficult to meet the needs of accurate identification of Solomon's seal germplasm resources, assessment of genetic diversity, and molecular-assisted breeding. This is particularly true for species like *Polygonatum yunnanensis*, which exhibits rich intraspecific variation and significant economic value, where there is a lack of molecular marker tools capable of effectively distinguishing different geographical populations or superior strains. Summary of the Invention

[0006] This invention addresses the aforementioned technical problems by developing 49 pairs of highly polymorphic EST-SSR markers based on combined rhizome transcriptome data from three medicinal Polygonatum species: *Polygonatum macrocephalum*, *Polygonatum yunnanensis*, and *Polygonatum multiflorum*. Nine core marker combinations were then selected from these. These marker combinations can accurately distinguish six closely related species: *Polygonatum macrocephalum*, *Polygonatum yunnanensis*, *Polygonatum multiflorum*, *Polygonatum macrocephalum*, *Polygonatum tuftense*, and *Polygonatum hubeiense*. Furthermore, they effectively differentiate the intraspecific geographic populations of *Polygonatum macrocephalum*. Simultaneously, fluorescently labeled capillary electrophoresis detection technology enables high-throughput, high-precision, and automated genotyping, providing an efficient molecular tool for the study of *Polygonatum* germplasm resources.

[0007] Through long-term exploration and experimentation, and continuous reform and innovation, the inventors have addressed the above-mentioned technical problems by providing an EST-SSR molecular marker primer combination for identifying plants of the genus *Polygonatum*. This primer combination includes at least one of the following nine primer pairs: (1) Primer pairs with nucleotide sequences as shown in SEQ ID NO.5-6; (2) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 9-10; (3) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 53-54; (4) Primer pairs with nucleotide sequences as shown in SEQ ID NO.3-4; (5) Primer pairs with nucleotide sequences as shown in SEQ ID NO.7-8; (6) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 63-64; (7) Primer pairs with nucleotide sequences as shown in SEQ ID NO.43-44; (8) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 67-68; (9) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 95-96; The nucleotide sequences of each primer pair were developed based on combined rhizome transcriptome data from three medicinal Polygonatum species: *P. kingianum* var. *grandifolium*, *P. kingianum*, and *P. cyrtonema*.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention, for the first time, develops EST-SSR markers based on combined rhizome transcriptome data from three medicinal Polygonatum species: *Polygonatum macrocephalum*, *Polygonatum yunnanensis*, and *Polygonatum multiflorum*. Compared to SSR markers from a single species, the obtained primers exhibit higher universality and conservation within the *Polygonatum* genus, with a high cross-species amplification success rate. Through large-scale screening and validation, 49 pairs of highly polymorphic EST-SSR markers (average PIC > 0.7) were obtained, and 9 core marker combinations were optimized and selected. These combinations can accurately distinguish six closely related species—*Polygonatum macrocephalum*, *Polygonatum yunnanensis*, *Polygonatum multiflorum*, *Polygonatum macrocephalum*, *Polygonatum tuftolens*, and *Polygonatum hubeiense*—in a single study. Furthermore, they effectively reveal the intraspecific genetic structure of *Polygonatum macrocephalum* in different geographical populations, solving the problems of incomplete species coverage and low intraspecific variation resolution of existing markers. Simultaneously, this invention employs fluorescently labeled capillary electrophoresis to replace traditional PAGE gel electrophoresis, achieving the ability to detect 2... The invention achieves precise resolution of differences in fragments larger than bp, boasts high throughput, high automation, and digitized results that are safe and non-toxic, overcoming the shortcomings of existing technologies such as low resolution, reliance on manual interpretation, and poor repeatability. The marker combinations of this invention are highly correlated with phyllotaxis type (irregular, whorled, alternate), and have the potential to identify more species in the genus *Polygonatum*. Furthermore, some markers are closely linked to genes related to carbohydrate metabolism, providing an efficient, precise, and economical molecular tool for functional genomics research of *Polygonatum* plants, construction of molecular identity cards for germplasm resources, protection of plant variety rights, and molecular-assisted breeding.

[0009] Preferably, the primer combination includes all nine pairs of primers.

[0010] Based on the above technical solution, the present invention can be further improved as follows: This invention also provides an EST-SSR molecular marker primer set for identifying plants of the genus *Polygonatum*, the primer set comprising the aforementioned primer combination, and supplementary primers selected from any one or more of the following primer pairs: (10) Primer pairs with nucleotide sequences as shown in SEQ ID NO.1-2; (11) Primer pairs with nucleotide sequences as shown in SEQ ID NO.11-12; (12) Primer pairs with nucleotide sequences as shown in SEQ ID NO.13-14; (13) Primer pairs with nucleotide sequences as shown in SEQ ID NO.15-16; (14) Primer pairs with nucleotide sequences as shown in SEQ ID NO.17-18; (15) Primer pairs with nucleotide sequences as shown in SEQ ID NO.19-20; (16) Primer pairs with nucleotide sequences as shown in SEQ ID NO.21-22; (17) Primer pairs with nucleotide sequences as shown in SEQ ID NO.23-24; (18) Primer pairs with nucleotide sequences as shown in SEQ ID NO.25-26; (19) Primer pairs with nucleotide sequences as shown in SEQ ID NO.27-28; (20) Primer pairs with nucleotide sequences as shown in SEQ ID NO.29-30; (21) Primer pairs with nucleotide sequences as shown in SEQ ID NO.31-32; (22) Primer pairs with nucleotide sequences as shown in SEQ ID NO.33-34; (23) Primer pairs with nucleotide sequences as shown in SEQ ID NO.35-36; (24) Primer pairs with nucleotide sequences as shown in SEQ ID NO.37-38; (25) Primer pairs with nucleotide sequences as shown in SEQ ID NO.39-40; (26) Primer pairs with nucleotide sequences as shown in SEQ ID NO.41-42; (27) Primer pairs with nucleotide sequences as shown in SEQ ID NO.45-46; (28) Primer pairs with nucleotide sequences as shown in SEQ ID NO.47-48; (29) Primer pairs with nucleotide sequences as shown in SEQ ID NO.49-50; (30) Primer pairs with nucleotide sequences as shown in SEQ ID NO.51-52; (31) Primer pairs with nucleotide sequences as shown in SEQ ID NO.55-56; (32) Primer pairs with nucleotide sequences as shown in SEQ ID NO.57-58; (33) Primer pairs with nucleotide sequences as shown in SEQ ID NO.59-60; (34) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 61-62; (35) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 65-66; (36) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 69-70; (37) Primer pairs with nucleotide sequences as shown in SEQ ID NO.71-72; (38) Primer pairs with nucleotide sequences as shown in SEQ ID NO.73-74; (39) Primer pairs with nucleotide sequences as shown in SEQ ID NO.75-76; (40) Primer pairs with nucleotide sequences as shown in SEQ ID NO.77-78; (41) Primer pairs with nucleotide sequences as shown in SEQ ID NO.79-80; (42) Primer pairs with nucleotide sequences as shown in SEQ ID NO.81-82; (43) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 83-84; (44) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 85-86; (45) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 87-88; (46) Primer pairs with nucleotide sequences as shown in SEQ ID NO.89-90; (47) Primer pairs with nucleotide sequences as shown in SEQ ID NO.91-92; (48) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 93-94; (49) Nucleotide sequences as shown in primer pairs in SEQ ID NO.97-98.

[0011] Furthermore, cluster analysis of the *Polygonatum* species using the primer set can classify the test samples into groups corresponding to phyllotaxis types; the phyllotaxis types include irregular phyllotaxis, whorled phyllotaxis, and alternate phyllotaxis.

[0012] The present invention also provides a kit for identifying plants of the genus *Polygonatum*, the kit comprising the aforementioned EST-SSR molecular marker primer combination or primer set.

[0013] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows: The kit prepared using the primer combination or primer set of this invention integrates 9 pairs of core EST-SSR markers and a fluorescent capillary electrophoresis detection system, enabling accurate, high-throughput, and automated identification of 6 closely related species of the genus Polygonatum and their intraspecific variations.

[0014] This invention also provides a method for identifying plants of the genus *Polygonatum*, comprising the following steps: (1) Extract genomic DNA from the samples of the *Polygonatum* species to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the EST-SSR molecular marker primer combination or primer set described above; (3) Capillary electrophoresis is performed on the amplification products of step (2) to obtain fragment size data of the amplification products; (4) Based on the fragment size data in step (3), species identification and / or genetic diversity analysis of plants in the genus Polygonatum are performed.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The identification method of this invention combines the EST-SSR marker combinations obtained through screening with fluorescent capillary electrophoresis detection technology. Through an optimized two-step PCR amplification procedure, it achieves efficient and accurate genotyping of *Polygonatum* DNA samples, overcoming the technical drawbacks of traditional PAGE gel electrophoresis, such as low resolution, low throughput, and reliance on manual interpretation. This method can rapidly acquire digital genotypic data, enabling efficient identification of six closely related species in the *Polygonatum* genus and detailed analysis of intraspecific variations in *Polygonatum yunnanensis*, providing a standardized technical process for the molecular identification and genetic evaluation of *Polygonatum* germplasm resources.

[0016] Based on the above technical solution, the present invention can be further improved as follows: Further: The PCR amplification program described in step (2) is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 65℃→55℃ gradient annealing for 30 s (decreasing by 1℃ per cycle), 72℃ extension for 40 s, run for 10 cycles; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, run for 25 cycles; 72℃ extension for 7 min.

[0017] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows: The two-step PCR amplification procedure combined with gradient annealing effectively improved the amplification success rate and specificity of multiple EST-SSR markers in complex templates, ensuring the accuracy and repeatability of subsequent capillary electrophoresis detection.

[0018] Based on the above technical solution, the present invention can be further improved as follows: Further: the species identification described in step (4) includes distinguishing at least two of the following: Polygonatum sibiricum, Polygonatum kingianum, Polygonatum cyrtonema, Polygonatum kingianum var. grandifolium, Polygonatum cirrhifolium, and Polygonatum zanlanscianense.

[0019] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows: This method expands the scope of species identification to six closely related species in the genus *Polygonatum*, and for the first time achieves efficient identification of species that are difficult to distinguish with existing markers, such as *Polygonatum yunnanensis*, *Polygonatum tuftolens*, and *Polygonatum hubeiense*, significantly improving the coverage and accuracy of germplasm resource identification in the genus *Polygonatum*.

[0020] Based on the above technical solution, the present invention can be further improved as follows: Furthermore, the genetic diversity analysis described in step (4) includes the analysis of intraspecific variation in different geographical populations of Polygonatum yunnanense.

[0021] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows: This method is the first to achieve precise differentiation of different geographical populations within the species *Polygonatum yunnanensis*, filling the gap in the existing ability of SSR markers to analyze variations within the *Polygonatum* genus. It provides key technical support for the accurate identification of germplasm resources, screening of core germplasm, and molecular marker-assisted breeding.

[0022] This invention also provides the application of the EST-SSR molecular marker primer combination or primer set, or the kit, in the identification of Solomon's seal germplasm resources, genetic diversity analysis, genetic map construction, or molecular marker-assisted breeding.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows: The application of the EST-SSR molecular marker primer combination, primer set and kit of the present invention in the identification of germplasm resources of Polygonatum, genetic diversity analysis, genetic map construction or molecular marker-assisted breeding provides a systematic solution for the accurate identification, genetic evaluation and breeding utilization of Polygonatum. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 This is a diagram showing the genetic analysis results of 21 Polygonatum germplasm resources based on 49 EST-SSR markers in an embodiment of the present invention. Figure 1In the table, A represents the UPGMA phylogenetic tree of 21 *Polygonatum* germplasm resources constructed based on 49 EST-SSR markers; B represents the principal coordinate analysis diagram of the 21 *Polygonatum* germplasm resources; and C represents the UPGMA phylogenetic tree of the 21 *Polygonatum* germplasm resources constructed based on 9 core EST-SSR markers. The analysis results show that the 21 germplasm resources were divided into three groups: Group I (irregular foliage, *Polygonatum yunnanensis*), Group II (whorled foliage, including *Polygonatum yunnanensis*, *Polygonatum*, *Polygonatum truncatum*, and *Polygonatum hubeiense*), and Group III (alternate foliage, *Polygonatum multiflorum*), which are highly correlated with foliage type.

[0026] Figure 2 This is a morphological characteristic diagram of four Polygonatum germplasm resources in Group II of this invention. Figure 2 In the image, A represents the fruit of *Polygonatum yunnanense*; B represents the red flower of *Polygonatum yunnanense*; C represents the yellowish-green flower of *Polygonatum yunnanense*; D represents the rhizome of *Polygonatum yunnanense*; E represents the rhizome of *Polygonatum*; F represents the flower of *Polygonatum yunnanense* (H58); G represents the flower of *Polygonatum yunnanense* (H14); H represents the fruit of *Polygonatum yunnanense*; I represents the immature fruit of *Polygonatum yunnanense*; J represents the mature fruit of *Polygonatum yunnanense*; K represents the flower of *Polygonatum yunnanense*; L represents the rhizome of *Polygonatum hubeiense*; and M represents the leaves and mature fruit of *Polygonatum hubeiense*.

[0027] Figure 3 Cluster analysis diagram of 21 Solomon's seal germplasm resources using the three EST-SSR markers FB-5, FB-9, and FB-49. The results showed that when using these three markers for clustering, Solomon's seal PK-3 was clustered within Solomon's seal macrophylla, and Solomon's seal H49 was clustered within Solomon's seal polyphylla, failing to achieve accurate species differentiation, demonstrating the limitations of using single or a small number of markers in the identification of Solomon's seal species.

[0028] Figure 4 Cluster analysis diagram of intraspecific resources of *Polygonatum yunnanensis* using the three EST-SSR markers FB-4, FB-8, and FB-9. The results show that when using these three markers for clustering, the intraspecific resources of *Polygonatum yunnanensis* do not have obvious geographical distribution characteristics and cannot distinguish intraspecific variation among different geographical populations, demonstrating the limitations of single or a small number of markers in intraspecific variation analysis. Detailed Implementation

[0029] The following description is based on specific embodiments.

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the present invention.

[0031] In this invention, unless otherwise specified, all equipment and raw materials are available from the market or commonly used in the industry. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art. Example 1

[0032] This example describes the development of EST-SSR markers and primer design.

[0033] 1.1 Plant materials The 21 accessions of *Polygonatum* species used in the experiment were collected from Sichuan, Hunan, Yunnan, and Chongqing provinces (details are shown in Table 1). All samples were cultivated at the Pidu Base of the Sichuan Academy of Agricultural Sciences. Fresh rhizome tissues were obtained from each sample, washed with water, dried, and stored at -80℃ for subsequent DNA extraction. Rhizomes of *Polygonatum kingianum* (PK), *Polygonatum cyrtonema* (PC), and *Polygonatum kingianum var. grandifolium* (PKG) were collected for subsequent RNA extraction and transcriptome sequencing. Three biological replicates were prepared for each rhizome tissue. Information on the 21 accessions is shown in Table 1.

[0034] Table 1 Germplasm resource information of 21 species of Polygonatum.

[0035] Note: "-" indicates an undetermined species.

[0036] 1.2 Transcriptome Sequencing and SSR Locus Mining Total RNA was extracted from the rhizomes of Polygonatum macrocephala, Polygonatum yunnanensis, and Polygonatum multiflorum using the CTAB method. mRNA was enriched using magnetic beads with Oligo(dT) ions. The enriched mRNA was fragmented and reverse transcribed to synthesize cDNA. Transcriptome sequencing libraries were constructed and high-throughput sequencing was performed on the Illumina sequencing platform.

[0037] After filtering the raw sequencing data to remove reads with adapters and low-quality reads, the Trinity software was used for sequence assembly, resulting in 198,752 unigenes with a total nucleotide length of 142,330,816 bp.

[0038] SSR sites in unigenes were identified using the microsatellite identification program MISA (http: / / pgrc.ipk-gatersleben.de / misa / ). The minimum repeat thresholds for different repeat units were set as follows: 10 repetitions for mononucleotides, 6 repetitions for dinucleotides, 5 repetitions for trinucleotides, 5 repetitions for tetranucleotides, 5 repetitions for pentanucleotides, and 5 repetitions for hexanucleotides. A compound SSR was defined as two SSR sequences spaced less than 100 bp apart.

[0039] The identification results showed that a total of 47,287 SSR motifs were identified, distributed across 36,842 unigenes containing SSR sites. SSR sites were classified into eight types: p1 (single nucleotide repeat), p2 (dinucleotide repeat), p3 (trinucleotide repeat), p4 (tetranucleotide repeat), p5 (pentanucleotide repeat), p6 (hexanucleotide repeat), c (compound repeat type), and c* (interrupted compound type). The statistical results of the SSR search are shown in Table 2, and the composition of SSR site types is shown in Table 3.

[0040] Table 2 Results of SSR search in the transcriptome

[0041] Table 3 Composition of SSR site types in the transcriptome

[0042] Note: p1, mononucleotide repeat type; p2, dinucleotide repeat type; p3, trinucleotide repeat type; p4, tetranucleotide repeat type; p5, pentanucleotide repeat type; p6, hexanucleotide repeat type; c, compound repeat type; c*, interrupted compound type.

[0043] 1.3 EST-SSR Primer Design and Synthesis Primers were designed using Primer3 software based on the flanking complementary sequences of the SSR loci. The parameters were set as follows: primer length approximately 20 bp, Tm value 57-63℃, and amplicon length 100-280 bp. Three primer pairs were designed for each predicted SSR locus.

[0044] Primers corresponding to 100 SSR sites were selected from 8 SSR types, with 20 pairs each for p2 and p3 types and 10 pairs for each of the other types. Before synthesis, the universal M13 adapter sequence (TGTAAAACGACGGCCAGT, SEQ ID NO.99) was added to the 5' end of each forward primer. Example 2

[0045] This embodiment describes the screening and verification of EST-SSR markers, based on embodiment 1.

[0046] 2.1 Genome extraction and quality testing Genomic DNA was extracted from 21 samples using the CTAB method: (1) Take about 100 mg of fresh rhizome tissue and grind it into powder in liquid nitrogen; (2) Add 800 μL of preheated CTAB extraction buffer, incubate in a 65°C water bath for 30 min, and invert and mix several times during the incubation period; (3) Add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently, and centrifuge at 12,000 rpm for 10 min; (4) Take out the supernatant, add an equal volume of pre-cooled isopropanol, and precipitate at -20℃ for 30 min; (5) Centrifuge at 12000 rpm for 10 min, discard the supernatant, and wash the precipitate twice with 70% ethanol; (6) After drying at room temperature, add 50 μL of TE buffer to dissolve the DNA and add RNase A to remove RNA contamination.

[0047] The purity and integrity of DNA were determined using a UV spectrophotometer and 1% agarose gel electrophoresis. DNA samples with clear and intact electrophoretic bands, where the OD260 / OD280 ratio was between 1.8 and 2.0, were used for subsequent experiments.

[0048] 2.2 PCR amplification PCR reaction system (10 μL): 2× Taq PCR Master Mix: 5 μL Forward primer (10 μM): 0.1 μL Reverse primer (10 μM): 0.3 μL M13 adapter primer (10 μM): 0.2 μL DNA template (30 ng / μL): 1 μL ddH2O: Add to a final volume of 10 μL PCR reaction procedure: Pre-denaturation at 94℃ for 5 minutes; First round of amplification (10 cycles): denaturation at 94℃ for 30 s, gradient annealing at 65℃→55℃ for 30 s (decreasing by 1℃ per cycle), extension at 72℃ for 40 s; Second round of amplification (25 cycles): denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 40 s; 72℃ extension: 7 min; Store at 4°C.

[0049] 2.3 Capillary electrophoresis detection The M13 primers were labeled with fluorescent dyes (FAM, HEX, TAMRA, ROX) to fluorescently label the PCR products. The capillary electrophoresis loading mixture contained: 0.3 μL of PCR product, 0.5 μL of molecular weight internal standard (LIZ500), and 9.5 μL of deionized formamide, and was added to a 96-well PCR plate. The mixture was denatured at 95°C for 5 min, immediately cooled on ice, centrifuged, and then 1×Buffer was added. Capillary electrophoresis was performed on an ABI 3730xl DNA sequencer for detection.

[0050] The electrophoresis results were analyzed using Gene Marker 2.2.0 software, and the amplified fragment size (bp) of each sample was recorded to build the original data matrix.

[0051] 2.4 Marking and filtering results One hundred primer pairs were validated in 21 Polygonatum samples, and the results showed that 49 primer pairs could amplify clear, stable, and polymorphic bands. Among the eight SSR types, the p2 type had the highest polymorphism rate (65%), while the c* type had the lowest polymorphism rate (20%). Primer information for the 49 polymorphic EST-SSR markers is shown in Table 4.

[0052] Table 4 Information on 49 pairs of polymorphic EST-SSR marker primers

[0053] Table 4. Information on 49 pairs of polymorphic EST-SSR marker primers (continued)

[0054] Note: Those marked with "*" are 9 pairs of core EST-SSR marker primers. Example 3

[0055] This example describes the genetic diversity analysis of the 49 EST-SSR markers screened in Example 2.

[0056] 3.1 Calculation of Genetic Diversity Parameters Using Popgene 1.32 and GenAlEx 6.5 software, genetic diversity parameters of 49 SSR loci were calculated, including: observed allele number (Na), effective allele number (Ne), Shannon information index (I), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC), inbreeding coefficient within subpopulation (Fis), overall inbreeding coefficient (Fit), genetic differentiation coefficient (Fst), and gene flow (Nm, calculated as Nm = 0.25×(1-Fst) / Fst).

[0057] The results showed that 49 SSR primer pairs amplified a total of 446 polymorphic alleles in 21 samples. The number of alleles (Na) ranged from 4 to 17, with an average of 9.10; the effective number of alleles (Ne) ranged from 1.55 to 10.00, with an average of 4.70; the Shannon Information Index (I) ranged from 0.70 to 2.48, with an average of 1.74; the observed heterozygosity (Ho) averaged 0.39, and the expected heterozygosity (He) averaged 0.76; the average polymorphism information content (PIC) was 0.718, with 47 primer pairs having a PIC value greater than 0.5, meeting the criteria for high polymorphism markers. The genetic differentiation coefficient (Fst) ranged from 0.0449 to 0.8054, and the average gene flow (Nm) was 0.876. Detailed genetic diversity parameters are shown in Table 5.

[0058] Table 5. Genetic diversity parameters of 49 SSR loci in Polygonatum germplasm resources

[0059] Table 5. Genetic diversity parameters of 49 SSR loci in Polygonatum germplasm resources (continued)

[0060] Note: Na, number of alleles; Ne, effective number of alleles; I, Shannon information index; PIC, polymorphism information content; Ho, observed heterozygosity; He, expected heterozygosity; Fish, inbreeding coefficient within a subpopulation; Fit, inbreeding coefficient of the total population; Fst, genetic differentiation coefficient; Nm, gene flow (calculated as Nm = 0.25 × (1-Fst) / Fst).

[0061] 3.2 Genetic Similarity and Cluster Analysis Using the UPGMA method in NTSYS-pc 2.10e software, a dendrogram was constructed based on the genetic similarity coefficients of 21 accessions. The coefficient values ​​ranged from 0.7159 to 0.9799, with a total variation of 0.264 and a mean of 0.7817. At the individual level, the highest value (0.9799) was found between accessions H50 and H54, indicating their closest kinship. Conversely, the lowest value (0.7159) was found between H1 and H70 / H72, reflecting the most distant kinship and significant genetic differentiation. When the genetic similarity coefficient was set to 0.75, the samples were divided into three groups (Group I, Group II, and Group III). Figure 1 (A)

[0062] Group I comprises 10 germplasm accessions, all originating from Sichuan Province, including cities such as Luzhou, Neijiang, Dazhou, Guang'an, Chengdu, and Nanchong. The average genetic similarity coefficient within this group is 0.88, with a minimum of 0.76, indicating a relatively homogeneous group. Germplasm in this group exhibits irregular leaf arrangement patterns and has been identified as *Polygonatum yunnanensis*. At a genetic similarity coefficient of 0.87, the resources in Group I can be further subdivided into three subgroups. The first subgroup consists of wild resources from Luzhou (Sichuan), which show significant genetic differentiation from all other resources. The second subgroup includes resources from Chengdu and southern Sichuan (Luzhou and Neijiang), including H11, H13, H26, and PKG-2. The third subgroup consists of resources from northeastern Sichuan (Dazhou, Guang'an, and Nanchong), including H16, H5, H50, H54, and H21. These results indicate that the *Polygonatum yunnanensis* germplasm resources exhibit a certain degree of regional distribution characteristics.

[0063] Group II consists of seven germplasms (H19, H44, H58, H71, H49, PK-3, and H70), with a wider geographical distribution, originating from four provinces and municipalities: Sichuan, Chongqing, Hunan, and Yunnan. The average genetic similarity coefficient is 0.78, with a minimum of 0.75. All germplasms in Group II exhibit whorled leaf arrangement and include species such as *Polygonatum yunnanense*, *Polygonatum sibiricum*, *Polygonatum sibiricum*, and *Polygonatum hubeiense*.

[0064] Group III comprises four accessions (H20, H73, H72, and PC-3), collected from Leshan and Shifang in Sichuan Province, and Xinhua in Hunan Province. Genetic similarity coefficients ranged from 0.80 to 0.84. All accessions in Group III exhibited alternate phyllotaxis and were identified as *Polygonatum multiflorum*. Furthermore, principal coordinate analysis was performed on 21 *Polygonatum* resources. Figure 1(B). The results were highly consistent with the cluster analysis results generated by NTSYS. Except for the highly similar H19 and H44, the resources in Group II showed significant morphological variation. Based on a genetic consistency coefficient of 0.80, this group could be further divided into four subgroups. In terms of flower morphology, size and color, H58 differed significantly from H19 and H44 (both are Polygonatum sibiricum PS). Compared with H9 (Polygonatum yunnanense, PK), H70 had smaller and dark green fruits, a nearly spherical rhizome, and a bitter taste, making it unsuitable for medicinal use. H49 was shorter, and its flower morphology, size and color were significantly different. H71 had smaller and dark green fruits. Combining morphological characteristics and molecular marker identification results, H58 and H71 were identified as Polygonatum multiflorum (PC), H49 as Polygonatum curlyleaf (PCI), and H70 as Polygonatum hubeiense (PZ). Figure 2 ). Example 4

[0065] This embodiment describes the screening and verification of the nine core EST-SSR tags described in Embodiment 3.

[0066] 4.1 Core Tag Filtering Strategy Based on the locus information and cluster analysis results of 49 polymorphic EST-SSR markers, the following strategy was used to screen core marker combinations: Species differentiation screening: Markers capable of independently distinguishing all six species of *Polygonatum* (*Polygonatum sibiricum*, *Polygonatum yunnanensis*, *Polygonatum multiflorum*, *Polygonatum yunnanensis*, *Polygonatum curcumum*, and *Polygonatum hubeiense*) were screened. Results showed that FB-5, FB-9, and FB-49 all possessed species differentiation capabilities. Functional annotation revealed that the three corresponding unigenes were all related to carbohydrate metabolism: FB-5 was annotated as a phosphoglucose mutase / phosphomannose mutase in the PFAM database, involved in glucose-mannose interconversion; FB-9 was annotated as a UDP-glucose-4-epimerase in the NT database, crucial for the synthesis of galactosylated cell wall precursors; and FB-49 was annotated as an α-galactosidase in the NR, NT, KO, SwissProt, and PFAM databases, involved in carbohydrate metabolism.

[0067] Screening for intraspecific variation differentiation: Markers capable of distinguishing different geographic populations within the species *Polygonatum yunnanensis* were screened. Results showed that seven markers—FB-4, FB-8, FB-9, FB-57, FB-42, FB-60, and FB-98—supported Group I subdivision. The first six markers were associated with carbohydrate metabolism, while FB-98 was associated with DNA repair. Database annotations indicated that the first six markers were associated with carbohydrate metabolism, while FB-98 was associated with DNA repair. Notably, the FB-9 marker could distinguish not only all six *Polygonatum* species but also intraspecific variation within *Polygonatum yunnanensis*. The gene associated with this marker encodes UDP-glucose-4-epimerase, an isomerase crucial for sugar metabolism and cell wall biosynthesis.

[0068] Deduplication and merging: Merge the above tags to remove duplicates and obtain 9 core EST-SSR tags: FB-5, FB-9, FB-49, FB-4, FB-8, FB-57, FB-42, FB-60, and FB-98.

[0069] 4.2 Validation of Core Tags Nine core markers were used to re-amplify PCR and detect the allele fragment size data of each sample using capillary electrophoresis. UPGMA cluster analysis was then performed using NTSYS-pc 2.10e software. Figure 1 (C)

[0070] The results showed that the nine core markers could effectively distinguish six species of *Polygonatum sibiricum*: *Polygonatum yunnanensis*, *Polygonatum multiflorum*, *Polygonatum yunnanensis*, *Polygonatum curcumum*, and *Polygonatum hubeiense*, and differentiate different geographical populations within the *Polygonatum yunnanensis* species. It is worth noting that... Figure 1 In the study, it was found that when the genetic similarity coefficient was 0.77, *Polygonatum yunnanense* and *Polygonatum macrocephalum* clustered together, indicating that this marker combination can reflect the close genetic relationship between *Polygonatum yunnanense* and *Polygonatum macrocephalum*.

[0071] Table 6. Marker information for 9 amplified SSR sites Example 5

[0072] This example describes the application of the nine core markers from Example 4 in the identification of Polygonatum species.

[0073] Nine pairs of core EST-SSR marker primers screened in Example 4 were used to identify the species of the Polygonatum sibiricum samples according to the PCR amplification and capillary electrophoresis detection methods in Example 2. The specific steps are as follows: (1) Extract genomic DNA from the Polygonatum sibiricum sample to be tested; (2) Using the extracted DNA as a template, PCR amplification was performed using 9 pairs of core EST-SSR marker primers. The PCR reaction system and procedure were the same as in Example 2. (3) The amplification products were subjected to fluorescently labeled capillary electrophoresis to obtain the size of each labeled amplification fragment; (4) Compare the amplification results of the sample to be tested with the allele data of the known species in Table 7, and determine the species affiliation of the sample to be tested based on the combination of characteristic bands.

[0074] Using the nine core EST-SSR marker combinations of this invention, the following six species of Polygonatum can be distinguished simultaneously in a single experiment: Polygonatum sibiricum Polygonatum kingianum Polygonatum cyrtonema Large-leaved Polygonatum kingianum var. grandifolium Polygonatum cirrhifolium Hubei Polygonatum (P. zanlanscianense) The marker combinations of this invention are highly correlated with phyllotaxis type (irregular phyllotaxis, whorled phyllotaxis, alternate phyllotaxis) and have the potential to identify more species of the genus *Polygonatum*. Example 6

[0075] This example describes the application of nine core markers in the intraspecific variation analysis of Polygonatum yunnanensis.

[0076] Nine pairs of core EST-SSR marker primers selected in Example 4 were used to conduct genetic diversity analysis and intraspecific variation studies on the germplasm resources of Polygonatum yunnanensis from different regions of Sichuan.

[0077] The samples include: wild resources in Luzhou (H1), resources in Chengdu (H11, H13, PKG-2), resources in southern Sichuan (Luzhou, Neijiang), and resources in northeastern Sichuan (Dazhou H16, Guang'an H5, Nanchong H50, H54, H21), etc.

[0078] PCR amplification and capillary electrophoresis were performed according to the method in Example 2 to obtain allele data for each sample. UPGMA cluster analysis was performed using NTSYS-pc 2.10e software.

[0079] The results show that ( Figure 1 The germplasm resources of *Polygonatum yunnanensis* (Group I) exhibit distinct regional distribution characteristics: Subgroup 1: Composed of wild resources from Luzhou, showing significant genetic differentiation from all other resources; Subgroup 2: Includes resources from Chengdu and southern Sichuan (Luzhou, Neijiang), including H11, H13, and PKG-2; The third subgroup includes resources from northeastern Sichuan (Dazhou, Guang'an, and Nanchong), including H16, H5, H50, H54, and H21.

[0080] The above results demonstrate that the nine core EST-SSR marker combinations of this invention can effectively reveal the intraspecific genetic structure of *Polygonatum yunnanensis*, distinguish germplasm resources from different geographical origins, and provide key technical support for germplasm resource identification, core germplasm screening, and molecular-assisted breeding of *Polygonatum yunnanensis*.

[0081] Comparative Example 1 During the marker selection process, it was found that a single marker or a few markers were insufficient to accurately distinguish all target species. For example, cluster analysis of 21 samples using three markers (FB-5, FB-9, and FB-49) showed that *Polygonatum yunnanense* PK-3 clustered within *Polygonatum macrocephalum*, and *Polygonatum truncatum* H49 clustered within *Polygonatum multiflorum*, failing to achieve accurate species differentiation. Figure 3 ).

[0082] The intraspecific resources of *Polygonatum yunnanensis* were classified using three markers: FB-4, FB-8, and FB-9. The results showed that the intraspecific resources did not have obvious geographical distribution characteristics, and it was impossible to distinguish the intraspecific variation among different geographical populations. Figure 4 ).

[0083] The above comparison shows that the nine core marker combinations of this invention achieve reliable identification of all six species and effective differentiation of intraspecific variations through allele combination patterns, making the scheme more robust. Example 7

[0084] This example describes the preparation and application of the reagent kit.

[0085] The identification kit for Polygonatum plants of the present invention comprises the following components: (1) EST-SSR labeled primer mixture: contains 9 pairs of core EST-SSR labeled primers (FB-5, FB-9, FB-49, FB-4, FB-8, FB-57, FB-42, FB-60, FB-98), with a concentration of 10 μM for each primer; (2) 2× PCR Master Mix: contains Taq DNA polymerase, dNTPs, Mg²⁺ and reaction buffer; (3) Positive control DNA: Genomic DNA of Polygonatum yunnanense (PKG-2); (4) Negative control: ; (5) Internal standard for molecular weight: LIZ500; (6) Deionized formamide; (7) Instruction manual.

[0086] Instructions for using the kit: Perform PCR amplification, capillary electrophoresis detection, and data analysis according to the methods described in Examples 2 and 5.

[0087] Application effects of the kit: Using this kit to identify unknown Polygonatum samples, it can accurately distinguish 6 species of Polygonatum in a single experiment, and can perform genetic diversity analysis on germplasm from different geographical sources of Polygonatum yunnanensis. The kit is easy to operate, the results are reliable and reproducible.

[0088] In the description of this invention, it should be understood that "-" and "~" represent a range between two values, and this range includes the endpoints. For example, "AB" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0089] In the description of this invention, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent three cases: A exists alone, A and B exist simultaneously, and B exists alone.

[0090] In the description of the invention, the numerical values ​​of time, temperature, ratio, and mass involved can be based on actual measurements, standard equipment parameters, simplified rounding results, or within an acceptable error range, ensuring the practicality and repeatability of the invention.

[0091] In the description of this invention, the terms “about” or “approximately” are used to express approximate values ​​or ranges, allowing for a certain degree of error to ensure the flexibility and practicality of the description, while remaining within an acceptable range of error, with the maximum error not exceeding 10% of the corresponding value or range.

[0092] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An EST-SSR molecular marker primer combination for identifying plants of the genus *Polygonatum*, characterized in that, The primer combination includes at least one of the following nine primer pairs: (1) Primer pairs with nucleotide sequences as shown in SEQ ID NO.5-6; (2) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 9-10; (3) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 53-54; (4) Primer pairs with nucleotide sequences as shown in SEQ ID NO.3-4; (5) Primer pairs with nucleotide sequences as shown in SEQ ID NO.7-8; (6) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 63-64; (7) Primer pairs with nucleotide sequences as shown in SEQ ID NO.43-44; (8) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 67-68; (9) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 95-96; The nucleotide sequences of each primer pair were developed based on combined rhizome transcriptome data from three medicinal Polygonatum species: *P. kingianum* var. *grandifolium*, *P. kingianum*, and *P. cyrtonema*.

2. The EST-SSR molecular marker primer combination according to claim 1, characterized in that, The primer combination includes all 9 pairs of primers.

3. An EST-SSR molecular marker primer set for identifying plants of the genus *Polygonatum*, characterized in that... The primer set comprises the primer combination as described in claim 1 or 2, and supplementary primers selected from any one or more of the following pairs of primers: (10) Primer pairs with nucleotide sequences as shown in SEQ ID NO.1-2; (11) Primer pairs with nucleotide sequences as shown in SEQ ID NO.11-12; (12) Primer pairs with nucleotide sequences as shown in SEQ ID NO.13-14; (13) Primer pairs with nucleotide sequences as shown in SEQ ID NO.15-16; (14) Primer pairs with nucleotide sequences as shown in SEQ ID NO.17-18; (15) Primer pairs with nucleotide sequences as shown in SEQ ID NO.19-20; (16) Primer pairs with nucleotide sequences as shown in SEQ ID NO.21-22; (17) Primer pairs with nucleotide sequences as shown in SEQ ID NO.23-24; (18) Primer pairs with nucleotide sequences as shown in SEQ ID NO.25-26; (19) Primer pairs with nucleotide sequences as shown in SEQ ID NO.27-28; (20) Primer pairs with nucleotide sequences as shown in SEQ ID NO.29-30; (21) Primer pairs with nucleotide sequences as shown in SEQ ID NO.31-32; (22) Primer pairs with nucleotide sequences as shown in SEQ ID NO.33-34; (23) Primer pairs with nucleotide sequences as shown in SEQ ID NO.35-36; (24) Primer pairs with nucleotide sequences as shown in SEQ ID NO.37-38; (25) Primer pairs with nucleotide sequences as shown in SEQ ID NO.39-40; (26) Primer pairs with nucleotide sequences as shown in SEQ ID NO.41-42; (27) Primer pairs with nucleotide sequences as shown in SEQ ID NO.45-46; (28) Primer pairs with nucleotide sequences as shown in SEQ ID NO.47-48; (29) Primer pairs with nucleotide sequences as shown in SEQ ID NO.49-50; (30) Primer pairs with nucleotide sequences as shown in SEQ ID NO.51-52; (31) Primer pairs with nucleotide sequences as shown in SEQ ID NO.55-56; (32) Primer pairs with nucleotide sequences as shown in SEQ ID NO.57-58; (33) Primer pairs with nucleotide sequences as shown in SEQ ID NO.59-60; (34) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 61-62; (35) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 65-66; (36) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 69-70; (37) Primer pairs with nucleotide sequences as shown in SEQ ID NO.71-72; (38) Primer pairs with nucleotide sequences as shown in SEQ ID NO.73-74; (39) Primer pairs with nucleotide sequences as shown in SEQ ID NO.75-76; (40) Primer pairs with nucleotide sequences as shown in SEQ ID NO.77-78; (41) Primer pairs with nucleotide sequences as shown in SEQ ID NO.79-80; (42) Primer pairs with nucleotide sequences as shown in SEQ ID NO.81-82; (43) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 83-84; (44) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 85-86; (45) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 87-88; (46) Primer pairs with nucleotide sequences as shown in SEQ ID NO.89-90; (47) Primer pairs with nucleotide sequences as shown in SEQ ID NO.91-92; (48) Primer pairs with nucleotide sequences as shown in SEQ ID NO. 93-94; (49) Nucleotide sequences as shown in primer pairs in SEQ ID NO.97-98.

4. The EST-SSR molecular marker primer set according to claim 3, characterized in that, Cluster analysis of plants in the genus *Polygonatum* using the primer set can classify the samples into groups corresponding to phyllotaxis types; the phyllotaxis types include irregular phyllotaxis, whorled phyllotaxis, and alternate phyllotaxis.

5. A kit for identifying plants of the genus *Polygonatum*, characterized in that, The kit contains the EST-SSR molecular marker primer combination or primer set as described in any one of claims 1-4.

6. A method for identifying plants of the genus *Polygonatum*, characterized in that, Includes the following steps: (1) Extract genomic DNA from the samples of the *Polygonatum* species to be tested; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the EST-SSR molecular marker primer combination or primer set described in any one of claims 1-4; (3) Capillary electrophoresis is performed on the amplification products of step (2) to obtain fragment size data of the amplification products; (4) Based on the fragment size data in step (3), species identification and / or genetic diversity analysis of plants in the genus Polygonatum are performed.

7. The method according to claim 6, characterized in that, The PCR amplification program described in step (2) is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 65℃→55℃ gradient annealing for 30 s (decreasing by 1℃ per cycle), 72℃ extension for 40 s, run for 10 cycles; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 40 s, run for 25 cycles; 72℃ extension for 7 min.

8. The method according to claim 6 or 7, characterized in that, The species identification described in step (4) includes distinguishing at least two of the following: Polygonatum sibiricum, Polygonatum kingianum, Polygonatum cyrtonema, Polygonatum kingianum var. grandifolium, Polygonatum cirrhifolium, and Polygonatum zanlanscianense.

9. The method according to claim 6 or 7, characterized in that, The genetic diversity analysis described in step (4) includes analyzing intraspecific variation in different geographical populations of Polygonatum yunnanense.

10. The application of the EST-SSR molecular marker primer combination or primer set according to any one of claims 1-4, or the kit according to claim 5, in the identification of germplasm resources of Polygonatum plants, genetic diversity analysis, genetic map construction, or molecular marker-assisted breeding.

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